This invention relates to blood fractionation and more particularly to the fractionation of blood coagulation factors with certain unique polyelectrolytes.
The process of blood coagulation is a complicated physiological activity that involves the interaction of numerous substances found in normal whole blood. It is known that certain factors associated with the blood coagulation mechanism are absent or seriously deficient in certain individuals. Thus, in those patients suffering from classical hemophilia, antihemophilic factor A (AHF, Factor VIII) is deficient. In those patients suffering from hemophilia B, plasma thromboplastin component (PTC, Factor IX) is missing from the blood. A small percentage of hemophiliacs also are lacking in the so-called Von Willebrand's Factor which is related to Factor VIII. Several other factors which are important in the coagulation mechanism, the absence of which can also lead to bleeding disorders, are, for example, Factors II, VII and X. The latter three factors together with Factor IX are frequently referred to as the prothrombin complex factors.
In the development of modern blood banking programs involving the collection and storage of large quantities of blood and blood components, the establishment of adequate preservation systems is critical. Since World War II it has been common practice to collect blood in a solution of citric acid, sodium citrate and dextrose, known as ACD blood. The problem of preserving blood is much simplified, however, when it is reduced to preservation of various blood components since it is easier to meet the environmental requirements of the separate components than of whole blood.
Moreover, it is wasteful and even detrimental to the patient to administer more blood components than required. Thus, the hemophiliac needing certain blood coagulation factors ideally should be given only those factors required or at least a purified concentrate of those factors.
The fractionation of blood coagulation factors, particularly Factor VIII and those factors associated with the prothrombin complex, is well-known, as can be seen from U.S. Pat. No. 3,682,881 and numerous other patents and publications. Various materials used in such fractionation are, for example, barium sulfate, aluminum hydroxide, polyethylene glycol, rivanol (6,9-diamino 2-ethoxyacridine lactate), glycine, DEAE-cellulose and DEAE-Sephadex.
Another group of substances which have been found useful in the fractionation of blood components are the water-insoluble, cross-linked polyelectrolyte copolymers described in U.S. Pat. No. 3,554,985. These substances are described as cross-linked copolymers of an (a) unsaturated monomer of 2 to 12 carbon atoms and (b) a monomer selected from the group consisting of (1) a mixture of an unsaturated polycarboxylic acid or anhydride and an unsaturated polycarboxylic acid amine-imide, and (2) an unsaturated polycarboxylic acid amine-imide, the polymeric units containing a defined minimum percentage of an amine-imide group which is a diloweralkylaminoloweralkylimide group wherein loweralkyl has 1 to 5 carbon atoms. Each of the polymeric units contains reactive sites in the form of an anhydride group or two carboxyl groups or derivatives of these groups. At least 3 percent of these reactive sites are said to be converted to the diloweralkylaminoloweralkylimide. The polyelectrolyte copolymers are thus generally only partially substituted with amine-imides.
The foregoing polyelectrolyte copolymers are known to be useful for preparation of various blood components such as albumin, gamma globulin, lipoproteins, hemoglobin and anti-trypsin factor as described in U.S. Pat. No. 3,555,001. In the fractionation of these components, the adsorption of a charged protein species to a charged insoluble substrate surface is accomplished through electrostatic interactions between sites of opposite charges, and this in turn is related to the isoelectric pH (IEpH) of the protein species and the pH of the medium.
In the fractionation of coagulation factors, such as Factor VIII, with certain of the polyelectrolytes described in U.S. Pat. Nos. 3,555,001 and 3,554,985, it has been found that use of the separated blood factor in standard clotting time tests causes a marked reduction in clotting time. This pre-activation of the separated Factor VIII component represents a serious deficiency in the clinical usefulness of this material. Such preactivation has also been observed by reaction in dogs when the separated Factor VIII was injected intravenously in the dogs. These reactions consisted of:
(1) a behavior change whereby the dog rotates in its cage for 24 hours,
(2) a hemolysis reaction evidenced by hemoglobinuria, and (3) a maked lowering of blood platelet count accompanied by rise in blood pressure.
This pre-activation has been found to be related to the structure and composition of the adsorbing polyelectrolyte in which there may be free hydroxyl groups capable of hydrogen bonding reactions. Thus, the polyelectrolyte copolymer may include the following polymeric structural units: ##STR1##
R=alkyl having from 1 to 5 carbon atoms
Z=bivalent hydrocarbon radical having from 2 to 18 carbon atoms
U.S. Pat. No. 3,555,001 discloses that the polyelectrolyte may possess from 2-100% amine-imide linkages, with the remaining carboxyl groups being in the anhydride form (col. 5, lines 29-31). It is further stated that the residual, non-modified polymer anhydride units may be converted to neutral groups or units by reaction of the unreacted anhydride units with compounds such as alkylamines, aminoalcohols and alcohols (col. 5, lines 54-60). In the illustrative formula, above, said partial substitution with the functional amine-imide likages is shown at I, the cross-linking imino-imide linkages are shown at III, and the remaining free carboxyl groups in the anhydride form are shown to be converted to neutral groups by blocking or reaction with an aminoalcohol at II thereby giving rise to hydroxyalkylimide units.
Notwithstanding the foregoing blocking of residual, free anhydride groups with aminoalcohol at II, it has been found that the free hydroxyls thus introduced on the polymer backbone in the form of hydroxyalkylimide groups as shown at II in the above formula contribute to the aforesaid pre-activation of Factor VIII which has been fractionated with this type of polyelectrolyte.
It has been further found that the pre-formation of the salt form of this type of polyelectrolyte, for example the hydrochloride salt, further accentuates the aforesaid pre-activation. Although the free amine form does not necessarily produce activation, the salt form of the polyelectrolyte is preferred from the standpoint of stability and the processing ability to remove water soluble extractable material which could find its way into the fractionated blood component.
Although the inventors are not bound by theory, it is believed that the above activation of Factor VIII may be due, in part, to the presence of plasma zymogens or enzymes resulting from zymogen activation in the separated Factor VIII fraction. These zymogens are known to be activated by negatively charged surfaces or hydroxylated surfaces capable of hydrogen bonding such as collagen. The aforesaid polymers having the free hydroxyl are capable of intra- and interchain hydrogen bonding with proteins with resultant areas of partial negative charge. In order to overcome this overall process of activation, the present inventors have synthesized new polyelectrolytes of the foregoing general type having partial substitution with amine-imides in which all the remaining free anhydrides are blocked with non-activating groups.